Of the 10 samples used, one was not usable (LV13) due to an error in sample prep. Another sample (LN11) was analyzed in triplicate to demonstrate the low level of variation between technical replicates. Data was normalized using internal positive controls and calculated in the nSolver software provided by nanostring. Housekeeping genes from the RNAseq data maintained a lower coefficient of variation then the 23 lactate correlation genes assessed. Of these 23 lactate correlation genes, only 12 yielded sufficient data above the false discovery threshold calculated using internal negative controls. Of these 12 genes, only 7 maintained a correlation of .8 with lactate levels as was observed in the RNAseq experiment.
| FN12 | FN11 | LV12 | FV13 | LV13 | LV11 | LN13 | FV12 | LN11.1 | LN11.2 | LN11.3 | FN13 | LN11 | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ACTB | 4990.04 | 7271.63 | 5190.07 | 3669.79 | 22 | 2786.80 | 3948.41 | 5873.95 | 5416.64 | 5196.70 | 5713.72 | 4314.44 | 5.442353e+03 |
| ALB | 243998.83 | 160622.86 | 186705.03 | 284541.81 | 22 | 93561.25 | 180669.25 | 220259.48 | 149218.75 | 141926.48 | 156008.36 | 184519.36 | 1.490512e+05 |
| ATG12 | 166.01 | 160.75 | 134.01 | 104.13 | 22 | 119.25 | 113.05 | 158.46 | 199.39 | 180.39 | 218.10 | 107.61 | 1.992933e+02 |
| ATG16L1 | 38.42 | 38.94 | 27.00 | 46.71 | 22 | 25.00 | 25.00 | 37.00 | 45.23 | 29.74 | 41.26 | 56.00 | 3.874333e+01 |
| ATG5 | 214.04 | 151.76 | 203.37 | 153.76 | 22 | 166.62 | 154.92 | 180.97 | 180.92 | 179.40 | 194.52 | 178.99 | 1.849467e+02 |
| ATP5ME | 2184.26 | 2035.82 | 2116.00 | 1673.84 | 22 | 1411.58 | 1916.63 | 2190.57 | 2334.47 | 2125.07 | 2322.43 | 2215.97 | 2.260657e+03 |
| ATP5MF | 1064.69 | 1023.40 | 1050.95 | 786.31 | 22 | 569.20 | 889.75 | 1178.57 | 1008.93 | 965.40 | 1016.80 | 1061.86 | 9.970433e+02 |
| BCL2 | 115.25 | 74.88 | 63.48 | 102.18 | 22 | 32.98 | 51.29 | 66.63 | 59.08 | 49.56 | 62.87 | 68.08 | 5.717000e+01 |
| BCL2L11 | 16.00 | 78.88 | 27.00 | 32.00 | 22 | 25.00 | 25.00 | 37.00 | 35.00 | 23.00 | 29.00 | 29.00 | 2.900000e+01 |
| CCL2 | 27.44 | 108.83 | 27.00 | 32.00 | 22 | 25.00 | 25.00 | 37.00 | 35.00 | 23.00 | 29.00 | 29.00 | 2.900000e+01 |
| CCL4 | 16.00 | 20.00 | 27.00 | 32.00 | 22 | 25.00 | 25.00 | 37.00 | 35.00 | 23.00 | 29.00 | 29.00 | 2.900000e+01 |
| CCL5 | 50.76 | 20.00 | 27.00 | 32.00 | 22 | 25.00 | 50.24 | 37.00 | 57.23 | 50.55 | 61.89 | 47.22 | 5.655667e+01 |
| COX17 | 499.42 | 469.27 | 460.82 | 257.89 | 22 | 312.93 | 363.23 | 543.82 | 438.46 | 336.01 | 429.32 | 487.56 | 4.012633e+02 |
| COX6B1 | 1355.56 | 1297.97 | 1168.50 | 864.17 | 22 | 811.94 | 924.30 | 1268.61 | 1390.16 | 1244.91 | 1377.34 | 1095.90 | 1.337470e+03 |
| COX7B | 1621.73 | 1975.91 | 1802.12 | 1067.56 | 22 | 1085.96 | 1430.93 | 1589.13 | 1487.08 | 1411.42 | 1550.25 | 1552.71 | 1.482917e+03 |
| CPD | 225.01 | 356.44 | 296.24 | 239.40 | 22 | 257.96 | 183.18 | 533.91 | 220.62 | 191.30 | 209.25 | 104.32 | 2.070567e+02 |
| CTH | 323.80 | 177.72 | 276.26 | 570.27 | 22 | 307.01 | 356.95 | 256.60 | 355.39 | 305.28 | 337.95 | 353.59 | 3.328733e+02 |
| CXCL10 | 26.07 | 20.00 | 27.00 | 32.00 | 22 | 25.00 | 31.40 | 37.00 | 35.00 | 23.00 | 29.00 | 29.00 | 2.900000e+01 |
| CYCS | 21.95 | 31.95 | 27.00 | 32.00 | 22 | 25.00 | 25.00 | 37.00 | 35.00 | 23.00 | 29.00 | 29.00 | 2.900000e+01 |
| CYP2B6 | 1551.76 | 60.90 | 742.95 | 373.69 | 22 | 138.71 | 13589.16 | 144.06 | 4499.09 | 4312.58 | 4553.49 | 18295.46 | 4.455053e+03 |
| DDIT3 | 159.15 | 136.79 | 150.47 | 119.70 | 22 | 67.66 | 96.30 | 99.94 | 118.15 | 122.90 | 121.82 | 121.89 | 1.209567e+02 |
| EPHX1 | 2004.52 | 755.82 | 1478.85 | 1300.14 | 22 | 796.71 | 5158.48 | 470.89 | 3637.86 | 3486.93 | 3854.01 | 6698.41 | 3.659600e+03 |
| ERO1A | 187.97 | 206.68 | 169.28 | 86.61 | 22 | 131.94 | 116.19 | 162.97 | 147.69 | 149.67 | 126.73 | 147.15 | 1.413633e+02 |
| G6PC1 | 1151.13 | 914.57 | 1260.19 | 1351.72 | 22 | 1568.05 | 748.44 | 1797.12 | 542.77 | 555.05 | 570.78 | 750.00 | 5.562000e+02 |
| GAPDH | 5857.16 | 7002.05 | 3898.14 | 4071.71 | 22 | 3197.00 | 4328.39 | 7301.92 | 6248.33 | 5709.13 | 6354.26 | 4523.08 | 6.103907e+03 |
| GPX2 | 1286.96 | 1028.39 | 1048.59 | 252.05 | 22 | 387.36 | 1831.85 | 460.08 | 2215.39 | 2092.36 | 2184.89 | 1580.17 | 2.164213e+03 |
| GSKIP | 200.32 | 224.65 | 181.04 | 185.87 | 22 | 146.32 | 129.80 | 192.68 | 130.15 | 136.78 | 149.33 | 96.63 | 1.387533e+02 |
| GSTA1 | 1702.68 | 578.10 | 1015.68 | 4037.65 | 22 | 416.12 | 9807.18 | 142.26 | 13434.51 | 12610.64 | 13825.52 | 7987.58 | 1.329022e+04 |
| GSTA2 | 841.05 | 32.95 | 297.42 | 632.56 | 22 | 25.00 | 6285.85 | 37.00 | 2702.78 | 2517.57 | 2735.04 | 3845.55 | 2.651797e+03 |
| HIF1A | 478.84 | 1180.15 | 285.66 | 188.79 | 22 | 198.76 | 244.94 | 378.15 | 382.16 | 328.08 | 353.67 | 220.72 | 3.546367e+02 |
| HRCT1 | 16.00 | 20.00 | 27.00 | 32.00 | 22 | 25.00 | 25.00 | 37.00 | 35.00 | 23.00 | 29.00 | 29.00 | 2.900000e+01 |
| HSP90B1 | 1909.85 | 2529.05 | 1749.22 | 241.34 | 22 | 1424.27 | 877.19 | 1358.64 | 884.31 | 854.39 | 997.15 | 1306.74 | 9.119500e+02 |
| HSPA5 | 408.86 | 316.51 | 293.89 | 47.68 | 22 | 187.76 | 126.66 | 161.16 | 123.69 | 106.06 | 125.75 | 284.41 | 1.185000e+02 |
| IFNG | 16.00 | 20.00 | 27.00 | 32.00 | 22 | 25.00 | 25.00 | 37.00 | 35.00 | 23.00 | 29.00 | 29.00 | 2.900000e+01 |
| IGHG3 | 87.81 | 20.00 | 27.00 | 32.00 | 22 | 25.00 | 86.88 | 37.00 | 155.08 | 143.72 | 151.29 | 74.67 | 1.500300e+02 |
| IL1B | 64.48 | 45.93 | 42.32 | 41.85 | 22 | 25.00 | 52.34 | 37.00 | 35.00 | 30.73 | 33.40 | 29.00 | 3.304333e+01 |
| IL6 | 16.00 | 20.00 | 27.00 | 32.00 | 22 | 25.00 | 25.00 | 37.00 | 35.00 | 23.00 | 29.00 | 29.00 | 2.900000e+01 |
| JUN | 120.74 | 159.75 | 116.38 | 111.91 | 22 | 46.52 | 309.84 | 101.74 | 234.46 | 236.89 | 267.22 | 282.21 | 2.461900e+02 |
| LDHA | 4132.53 | 4620.78 | 3273.92 | 1574.58 | 22 | 2889.14 | 1486.41 | 4193.88 | 1852.62 | 1696.88 | 1869.53 | 1997.45 | 1.806343e+03 |
| MAP1LC3A | 105.65 | 52.92 | 55.25 | 94.40 | 22 | 56.67 | 73.27 | 69.33 | 69.23 | 63.43 | 96.28 | 94.44 | 7.631333e+01 |
| MCL1 | 255.20 | 284.56 | 280.96 | 239.40 | 22 | 178.46 | 264.83 | 232.29 | 397.85 | 354.84 | 395.91 | 259.15 | 3.828667e+02 |
| MZB1 | 16.00 | 20.00 | 27.00 | 32.00 | 22 | 25.00 | 25.00 | 37.00 | 35.00 | 23.00 | 29.00 | 29.00 | 2.900000e+01 |
| NDUFA13 | 524.11 | 562.12 | 565.44 | 392.18 | 22 | 353.53 | 479.42 | 594.24 | 611.08 | 566.95 | 641.52 | 564.42 | 6.065167e+02 |
| NDUFS6 | 1008.44 | 867.64 | 911.05 | 572.22 | 22 | 569.20 | 715.99 | 986.79 | 846.46 | 798.88 | 883.19 | 767.57 | 8.428433e+02 |
| NDUFS8 | 552.92 | 540.16 | 463.17 | 341.58 | 22 | 325.62 | 347.53 | 544.72 | 400.62 | 373.67 | 430.30 | 434.85 | 4.015300e+02 |
| NKG7 | 24.70 | 23.96 | 27.00 | 32.00 | 22 | 25.00 | 52.34 | 37.00 | 42.46 | 41.63 | 39.30 | 29.00 | 4.113000e+01 |
| NLRP3 | 16.00 | 20.00 | 27.00 | 32.00 | 22 | 25.00 | 25.00 | 37.00 | 35.00 | 23.00 | 29.00 | 29.00 | 2.900000e+01 |
| NRF1 | 26.07 | 23.96 | 27.00 | 32.00 | 22 | 25.00 | 52.34 | 37.00 | 38.77 | 28.74 | 52.07 | 66.98 | 3.986000e+01 |
| OAZ1 | 1446.11 | 1067.33 | 1175.55 | 704.57 | 22 | 993.78 | 860.44 | 1385.65 | 1355.08 | 1186.43 | 1339.03 | 1124.46 | 1.293513e+03 |
| PCK1 | 4771.89 | 2575.97 | 3594.84 | 4313.05 | 22 | 7782.75 | 2273.58 | 6369.14 | 2136.93 | 2016.04 | 2197.66 | 4848.11 | 2.116877e+03 |
| PCSK6 | 1002.95 | 1087.30 | 1458.86 | 781.45 | 22 | 1005.62 | 916.97 | 1505.40 | 858.46 | 845.47 | 890.07 | 915.82 | 8.646667e+02 |
| PMAIP1 | 16.00 | 20.00 | 27.00 | 32.00 | 22 | 25.00 | 25.00 | 37.00 | 35.00 | 23.00 | 29.00 | 29.00 | 2.900000e+01 |
| PPARGC1A | 104.27 | 87.86 | 195.14 | 165.44 | 22 | 299.40 | 102.58 | 152.16 | 84.92 | 84.25 | 99.22 | 133.97 | 8.946333e+01 |
| PPBP | 16.00 | 20.00 | 27.00 | 32.00 | 22 | 25.00 | 25.00 | 37.00 | 35.00 | 23.00 | 29.00 | 29.00 | 2.900000e+01 |
| PRF1 | 16.00 | 20.00 | 27.00 | 32.00 | 22 | 25.00 | 26.17 | 37.00 | 35.00 | 23.00 | 29.00 | 29.00 | 2.900000e+01 |
| PZP | 2438.08 | 3867.95 | 8327.63 | 1415.95 | 22 | 5873.00 | 4034.25 | 6594.23 | 6687.72 | 6336.54 | 7001.67 | 6948.78 | 6.675310e+03 |
| RPL13A | 1406.32 | 1940.97 | 1511.76 | 1305.98 | 22 | 933.73 | 1480.13 | 1499.10 | 2161.85 | 2045.77 | 2129.87 | 1492.32 | 2.112497e+03 |
| SERF2 | 28.81 | 20.00 | 27.00 | 32.00 | 22 | 25.00 | 29.31 | 37.00 | 41.54 | 31.72 | 38.31 | 35.14 | 3.719000e+01 |
| SOCS3 | 50.76 | 72.89 | 48.20 | 32.00 | 22 | 54.97 | 40.82 | 71.13 | 37.85 | 38.66 | 35.37 | 29.00 | 3.729333e+01 |
| SQSTM1 | 1881.04 | 2145.65 | 2741.39 | 1218.40 | 22 | 1518.15 | 2393.96 | 1411.76 | 5312.33 | 4886.46 | 5420.96 | 3088.96 | 5.206583e+03 |
| TFAM | 227.76 | 152.76 | 181.04 | 249.13 | 22 | 117.56 | 174.81 | 216.99 | 180.92 | 145.70 | 181.75 | 183.38 | 1.694567e+02 |
| TKT | 223.64 | 223.65 | 102.27 | 111.91 | 22 | 75.27 | 277.39 | 129.65 | 390.46 | 377.64 | 364.48 | 453.52 | 3.775267e+02 |
| TMEM63A | 193.45 | 184.71 | 114.03 | 98.29 | 22 | 128.56 | 38.73 | 243.10 | 62.77 | 65.42 | 66.80 | 105.42 | 6.499667e+01 |
| TMEM92 | 16.00 | 20.00 | 27.00 | 32.00 | 22 | 25.00 | 25.00 | 37.00 | 43.38 | 41.63 | 29.00 | 29.00 | 3.800333e+01 |
| TNF | 16.00 | 20.00 | 27.00 | 32.00 | 22 | 25.00 | 25.00 | 37.00 | 35.00 | 23.00 | 29.00 | 29.00 | 2.900000e+01 |
| TRBC1 | 34.30 | 20.97 | 27.00 | 32.00 | 22 | 25.00 | 30.36 | 37.00 | 35.00 | 28.74 | 31.44 | 29.00 | 3.172667e+01 |
| WARS1 | 171.50 | 216.66 | 116.38 | 95.37 | 22 | 74.43 | 128.75 | 133.25 | 151.39 | 112.99 | 145.40 | 169.11 | 1.365933e+02 |
| XBP1 | 2819.50 | 2797.63 | 3331.52 | 1832.46 | 22 | 2391.83 | 2180.42 | 4812.42 | 4172.32 | 4008.29 | 4134.98 | 2420.21 | 4.105197e+03 |
| ZNF746 | 16.00 | 22.96 | 27.00 | 32.00 | 22 | 25.00 | 43.96 | 37.00 | 37.85 | 23.00 | 31.44 | 45.02 | 3.076333e+01 |
| NEG_A | 7.00 | 17.00 | 12.00 | 11.00 | 22 | 10.00 | 11.00 | 13.00 | 10.00 | 11.00 | 12.00 | 10.00 | 1.100000e+01 |
| NEG_B | 9.00 | 20.00 | 12.00 | 30.00 | 7 | 14.00 | 20.00 | 23.00 | 24.00 | 21.00 | 20.00 | 17.00 | 2.166667e+01 |
| NEG_C | 1.00 | 2.00 | 2.00 | 2.00 | 2 | 7.00 | 4.00 | 7.00 | 4.00 | 2.00 | 7.00 | 5.00 | 4.333333e+00 |
| NEG_D | 1.00 | 7.00 | 4.00 | 6.00 | 6 | 3.00 | 7.00 | 4.00 | 4.00 | 7.00 | 4.00 | 2.00 | 5.000000e+00 |
| NEG_E | 11.00 | 20.00 | 10.00 | 10.00 | 10 | 14.00 | 12.00 | 14.00 | 22.00 | 15.00 | 25.00 | 21.00 | 2.066667e+01 |
| NEG_F | 16.00 | 20.00 | 27.00 | 30.00 | 21 | 25.00 | 25.00 | 37.00 | 35.00 | 23.00 | 29.00 | 29.00 | 2.900000e+01 |
| NEG_G | 1.00 | 2.00 | 1.00 | 2.00 | 1 | 2.00 | 2.00 | 1.00 | 1.00 | 3.00 | 4.00 | 1.00 | 2.666667e+00 |
| NEG_H | 6.00 | 5.00 | 7.00 | 32.00 | 1 | 1.00 | 3.00 | 15.00 | 4.00 | 3.00 | 5.00 | 6.00 | 4.000000e+00 |
| POS_A | 12645.00 | 17909.00 | 15985.00 | 18718.00 | 23104 | 22473.00 | 18006.00 | 20221.00 | 20055.00 | 19011.00 | 20336.00 | 16846.00 | 1.980067e+04 |
| POS_B | 4673.00 | 6644.00 | 6209.00 | 6811.00 | 8120 | 8083.00 | 6495.00 | 7506.00 | 7123.00 | 6886.00 | 7208.00 | 6200.00 | 7.072333e+03 |
| POS_C | 1229.00 | 1807.00 | 1596.00 | 1888.00 | 2163 | 2294.00 | 1762.00 | 1950.00 | 2004.00 | 1944.00 | 1960.00 | 1662.00 | 1.969333e+03 |
| POS_D | 277.00 | 404.00 | 353.00 | 435.00 | 473 | 471.00 | 391.00 | 454.00 | 456.00 | 423.00 | 469.00 | 379.00 | 4.493333e+02 |
| POS_E | 93.00 | 122.00 | 83.00 | 111.00 | 93 | 93.00 | 104.00 | 140.00 | 117.00 | 108.00 | 105.00 | 94.00 | 1.100000e+02 |
| POS_F | 53.00 | 63.00 | 54.00 | 67.00 | 74 | 99.00 | 60.00 | 66.00 | 70.00 | 60.00 | 52.00 | 61.00 | 6.066667e+01 |
* Positive Controls: Each mRNA Expression CodeSet contains
probes designed against fourteen ERCC transcript sequences. Six of these
sequences are used as positive hybridization controls and eight are
designed as negative controls. For each positive control, in vitro
transcribed RNA targets are pre-mixed with the Reporter CodeSet during
manufacturing. Six different transcripts are provided, each at one of
the following concentrations in the 30 μL hybridization reaction: 128
fM, 32 fM, 8 fM, 2 fM, 0.5 fM, and 0.125 fM. This range of
concentrations corresponds to the expression levels of most mRNAs of
interest present in 100 ng of total RNA.
IMPORTANT
Positive controls only account for variation hybridizaiton and recovery
efficiency between lanes, not differences in total mRNA loaded!
* Negative Controls: NanoString includes several probes in
each CodeSet for which no target is expected to be present. These
negative controls can be used to estimate background values in your
experiment, but may not capture minor probe-specific differences in
background for every probe in a CodeSet.
Excluding any probes that did not exceed the maximum count observed in the negative controls for that sample. This is the most stringent false discovery identification method suggested by nanostring
Housekeeping genes should have generally lower coefficients of variation compared to the lactate correlation genes
Excluding any gene that does not have a correlation of at least .8 between the nanostring data and 3-hour lactate levels
Note: GSTA1 is only able to meet the cutoff when rounded. (r=
0.7977059)
There are 7 genes that meet the correlation cutoff of .8 that
was previously used with the RNAseq data results. All of these genes
positively correlated with lactate, with no negatively correlating genes
having sufficient levels of correlation. Alternatively, setting the
cutoff more leniently to .65 would still only yield a total of 10 genes
which would include a single negatively correlating gene